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MicroRNA-762 is upregulated in human corneal epithelial cells in response to tear fluid and Pseudomonas aeruginosa antigens and negatively regulates the expression of host defense genes encoding RNase7 and ST2.

Mun J, Tam C, Chan G, Kim JH, Evans D, Fleiszig S - PLoS ONE (2013)

Bottom Line: Without tears or antigens, an antagomir reduced miR-762 expression relative to scrambled controls by ∼50%, increased expression of genes encoding RNase7 (∼80 %), ST2 (∼58%) and Rab5a (∼75%), without affecting P. aeruginosa internalization.Tear fluid alone also induced miR-762 expression ∼ 4-fold, which was reduced by the miR-762 antagomir.Since RNase7 and ST2 inhibit P. aeruginosa internalization, and are upregulated by tear fluid, other tear-induced mechanisms must counteract inhibitory effects of miR-762 to regulate resistance to bacteria.

View Article: PubMed Central - PubMed

Affiliation: School of Optometry, University of California, Berkeley, California, United States of America.

ABSTRACT
Mucosal surfaces regulate defenses against infection and excessive inflammation. We previously showed that human tears upregulated epithelial expression of genes encoding RNase7 and ST2, which inhibited Pseudomonas aeruginosa invasion of human corneal epithelial cells. Here, microRNA microarrays were used to show that a combination of tear fluid exposure (16 h) then P. aeruginosa antigens (3 h) upregulated miR-762 and miR-1207, and down-regulated miR-92 and let-7b (all > 2-fold) in human corneal epithelial cells compared to P. aeruginosa antigens alone. RT-PCR confirmed miR-762 upregulation ∼ 3-fold in tear-antigen exposed cells. Without tears or antigens, an antagomir reduced miR-762 expression relative to scrambled controls by ∼50%, increased expression of genes encoding RNase7 (∼80 %), ST2 (∼58%) and Rab5a (∼75%), without affecting P. aeruginosa internalization. However, P. aeruginosa invasion was increased > 3-fold by a miR-762 mimic which reduced RNase7 and ST2 gene expression. Tear fluid alone also induced miR-762 expression ∼ 4-fold, which was reduced by the miR-762 antagomir. Combination of tear fluid and miR-762 antagomir increased RNase7 and ST2 gene expression. These data show that mucosal fluids, such as tears, can modulate epithelial microRNA expression to regulate innate defense genes, and that miR-762 negatively regulates RNase7, ST2 and Rab5a genes. Since RNase7 and ST2 inhibit P. aeruginosa internalization, and are upregulated by tear fluid, other tear-induced mechanisms must counteract inhibitory effects of miR-762 to regulate resistance to bacteria. These data also suggest a complex relationship between tear induction of miR-762, its modulation of innate defense genes, and P. aeruginosa internalization.

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Upregulation of miR-762 in human corneal epithelial cells in response to human tear fluid and P. aeruginosa antigens.a Microarray analysis of human corneal epithelial microRNA expression in response to P. aeruginosa antigens (3 h incubation) with and without prior exposure to human tear fluid for 16 h (see Material and Methods). Red and blue boxes indicate microRNAs which were up- or down-regulated respectively by 2-fold or more in response to bacterial antigens with prior tear fluid exposure (expressed relative to bacterial antigen challenge without prior tear exposure). The y-axis is shown as log2 (comparing tear fluid + antigens versus media + antigens) with a value of 1 or -1 meaning that the test group was up- or down-regulated respectively by 2 fold. The x-axis shows mean values of the test and control and represent raw data values from the microarray. Only values greater than 5 were considered. Those below 5 were below detection (i.e. considered background). Since there were multiple data points between 3–4 and 6–7 the software generated the figure to distribute these data points along the x-axis. PA  =  P. aeruginosa antigens, Media  =  High Calcium KGM-2. Eight wells of hTCEpi (grown in 96-well plates) were pooled to obtain sufficient RNA for each treatment group, b Real-time PCR confirmed a ∼3-fold upregulation of miR-762 in tear-treated epithelial cells in response to antigenic challenge (* Significant difference vs. control, t-Test).
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pone-0057850-g001: Upregulation of miR-762 in human corneal epithelial cells in response to human tear fluid and P. aeruginosa antigens.a Microarray analysis of human corneal epithelial microRNA expression in response to P. aeruginosa antigens (3 h incubation) with and without prior exposure to human tear fluid for 16 h (see Material and Methods). Red and blue boxes indicate microRNAs which were up- or down-regulated respectively by 2-fold or more in response to bacterial antigens with prior tear fluid exposure (expressed relative to bacterial antigen challenge without prior tear exposure). The y-axis is shown as log2 (comparing tear fluid + antigens versus media + antigens) with a value of 1 or -1 meaning that the test group was up- or down-regulated respectively by 2 fold. The x-axis shows mean values of the test and control and represent raw data values from the microarray. Only values greater than 5 were considered. Those below 5 were below detection (i.e. considered background). Since there were multiple data points between 3–4 and 6–7 the software generated the figure to distribute these data points along the x-axis. PA  =  P. aeruginosa antigens, Media  =  High Calcium KGM-2. Eight wells of hTCEpi (grown in 96-well plates) were pooled to obtain sufficient RNA for each treatment group, b Real-time PCR confirmed a ∼3-fold upregulation of miR-762 in tear-treated epithelial cells in response to antigenic challenge (* Significant difference vs. control, t-Test).

Mentions: Microarray analysis was used to compare microRNA expression in human corneal epithelial cells after 16 h exposure to undiluted human tear fluid or cell culture media (control), and 3 h exposure to bacterial antigens (see Methods). Very few corneal epithelial microRNAs were up or down-regulated (2 and 6 probe sets, respectively) by 2-fold or more in tear and bacterial antigen treated cells relative to controls (Fig. 1 a). MiR-762 and miR-1207 were both significantly upregulated; miR-92 and let-7b were both significantly downregulated. MiR-762 showed greatest upregulation in tear and bacterial antigen treated cells, which was confirmed by RT-PCR (> 3-fold, Fig. 1 b, p < 0.05, t-Test). These microarray data have been deposited in NCBI's Gene Expression Omnibus [31], and are accessible through GEO Series accession number GSE39341. ().


MicroRNA-762 is upregulated in human corneal epithelial cells in response to tear fluid and Pseudomonas aeruginosa antigens and negatively regulates the expression of host defense genes encoding RNase7 and ST2.

Mun J, Tam C, Chan G, Kim JH, Evans D, Fleiszig S - PLoS ONE (2013)

Upregulation of miR-762 in human corneal epithelial cells in response to human tear fluid and P. aeruginosa antigens.a Microarray analysis of human corneal epithelial microRNA expression in response to P. aeruginosa antigens (3 h incubation) with and without prior exposure to human tear fluid for 16 h (see Material and Methods). Red and blue boxes indicate microRNAs which were up- or down-regulated respectively by 2-fold or more in response to bacterial antigens with prior tear fluid exposure (expressed relative to bacterial antigen challenge without prior tear exposure). The y-axis is shown as log2 (comparing tear fluid + antigens versus media + antigens) with a value of 1 or -1 meaning that the test group was up- or down-regulated respectively by 2 fold. The x-axis shows mean values of the test and control and represent raw data values from the microarray. Only values greater than 5 were considered. Those below 5 were below detection (i.e. considered background). Since there were multiple data points between 3–4 and 6–7 the software generated the figure to distribute these data points along the x-axis. PA  =  P. aeruginosa antigens, Media  =  High Calcium KGM-2. Eight wells of hTCEpi (grown in 96-well plates) were pooled to obtain sufficient RNA for each treatment group, b Real-time PCR confirmed a ∼3-fold upregulation of miR-762 in tear-treated epithelial cells in response to antigenic challenge (* Significant difference vs. control, t-Test).
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Related In: Results  -  Collection

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pone-0057850-g001: Upregulation of miR-762 in human corneal epithelial cells in response to human tear fluid and P. aeruginosa antigens.a Microarray analysis of human corneal epithelial microRNA expression in response to P. aeruginosa antigens (3 h incubation) with and without prior exposure to human tear fluid for 16 h (see Material and Methods). Red and blue boxes indicate microRNAs which were up- or down-regulated respectively by 2-fold or more in response to bacterial antigens with prior tear fluid exposure (expressed relative to bacterial antigen challenge without prior tear exposure). The y-axis is shown as log2 (comparing tear fluid + antigens versus media + antigens) with a value of 1 or -1 meaning that the test group was up- or down-regulated respectively by 2 fold. The x-axis shows mean values of the test and control and represent raw data values from the microarray. Only values greater than 5 were considered. Those below 5 were below detection (i.e. considered background). Since there were multiple data points between 3–4 and 6–7 the software generated the figure to distribute these data points along the x-axis. PA  =  P. aeruginosa antigens, Media  =  High Calcium KGM-2. Eight wells of hTCEpi (grown in 96-well plates) were pooled to obtain sufficient RNA for each treatment group, b Real-time PCR confirmed a ∼3-fold upregulation of miR-762 in tear-treated epithelial cells in response to antigenic challenge (* Significant difference vs. control, t-Test).
Mentions: Microarray analysis was used to compare microRNA expression in human corneal epithelial cells after 16 h exposure to undiluted human tear fluid or cell culture media (control), and 3 h exposure to bacterial antigens (see Methods). Very few corneal epithelial microRNAs were up or down-regulated (2 and 6 probe sets, respectively) by 2-fold or more in tear and bacterial antigen treated cells relative to controls (Fig. 1 a). MiR-762 and miR-1207 were both significantly upregulated; miR-92 and let-7b were both significantly downregulated. MiR-762 showed greatest upregulation in tear and bacterial antigen treated cells, which was confirmed by RT-PCR (> 3-fold, Fig. 1 b, p < 0.05, t-Test). These microarray data have been deposited in NCBI's Gene Expression Omnibus [31], and are accessible through GEO Series accession number GSE39341. ().

Bottom Line: Without tears or antigens, an antagomir reduced miR-762 expression relative to scrambled controls by ∼50%, increased expression of genes encoding RNase7 (∼80 %), ST2 (∼58%) and Rab5a (∼75%), without affecting P. aeruginosa internalization.Tear fluid alone also induced miR-762 expression ∼ 4-fold, which was reduced by the miR-762 antagomir.Since RNase7 and ST2 inhibit P. aeruginosa internalization, and are upregulated by tear fluid, other tear-induced mechanisms must counteract inhibitory effects of miR-762 to regulate resistance to bacteria.

View Article: PubMed Central - PubMed

Affiliation: School of Optometry, University of California, Berkeley, California, United States of America.

ABSTRACT
Mucosal surfaces regulate defenses against infection and excessive inflammation. We previously showed that human tears upregulated epithelial expression of genes encoding RNase7 and ST2, which inhibited Pseudomonas aeruginosa invasion of human corneal epithelial cells. Here, microRNA microarrays were used to show that a combination of tear fluid exposure (16 h) then P. aeruginosa antigens (3 h) upregulated miR-762 and miR-1207, and down-regulated miR-92 and let-7b (all > 2-fold) in human corneal epithelial cells compared to P. aeruginosa antigens alone. RT-PCR confirmed miR-762 upregulation ∼ 3-fold in tear-antigen exposed cells. Without tears or antigens, an antagomir reduced miR-762 expression relative to scrambled controls by ∼50%, increased expression of genes encoding RNase7 (∼80 %), ST2 (∼58%) and Rab5a (∼75%), without affecting P. aeruginosa internalization. However, P. aeruginosa invasion was increased > 3-fold by a miR-762 mimic which reduced RNase7 and ST2 gene expression. Tear fluid alone also induced miR-762 expression ∼ 4-fold, which was reduced by the miR-762 antagomir. Combination of tear fluid and miR-762 antagomir increased RNase7 and ST2 gene expression. These data show that mucosal fluids, such as tears, can modulate epithelial microRNA expression to regulate innate defense genes, and that miR-762 negatively regulates RNase7, ST2 and Rab5a genes. Since RNase7 and ST2 inhibit P. aeruginosa internalization, and are upregulated by tear fluid, other tear-induced mechanisms must counteract inhibitory effects of miR-762 to regulate resistance to bacteria. These data also suggest a complex relationship between tear induction of miR-762, its modulation of innate defense genes, and P. aeruginosa internalization.

Show MeSH
Related in: MedlinePlus